The accelerated pubertal timing, premature anestrus, and ovarian malformations induced by neonatal exposure to BPA could result from disrupted organization anywhere within the HPG axis, including the hypothalamus, ovary, and/or pituitary gland. The exposure was specifically timed to target the hypothalamus. Our results do not support the hypothesis that BPA can defeminize the hypothalamus, a process in which, as we have shown previously [11
], ESR1 appears to play a significant mechanistic role. Instead, GnRH neurons seem to retain the capacity to respond (by producing FOS) to hormone priming by estrogen and progesterone. These results suggest that steroid-positive feedback was not defeminized by BPA but do not rule out the possibility, however, that other signaling mechanisms on GnRH neurons could have been altered.
Effects through ESR1 (by PPT or other endocrine disruptors) on GnRH are most likely indirect, because ESR1 is not known to be present in GnRH neurons. Instead, disruption likely occurs within ESR1-containing neurons in the AVPV, the arcuate nucleus (ARC), or elsewhere, which send projections to GnRH neurons [23
]. This may include the recently discovered population of neurons in the AVPV that express the peptide kisspeptin and send afferents to GnRH neurons [15
]. The vast majority of kisspeptin neurons in this region coexpress ESR1, and the administration of kisspeptin can induce early puberty in adolescent animals and generate a GnRH surge in ovariectomized animals [53
]. A recent study by Navarro et al. [55
] found reductions in hypothalamic Kiss1 mRNA levels after neonatal BPA exposure at doses higher than those used here. We subsequently determined that the density of hypothalamic kisspeptin-immunoreactive fibers is reduced by the high, but not the low, dose of BPA used in the present study, and that the effect was more pronounced in the ARC than the AVPV [43
]. Collectively, these observations support the hypothesis that signaling pathways on GnRH neurons may be altered by developmental exposure to BPA and that steroid-negative (rather than positive) feedback may be impaired.
Even if GnRH release following estrogen administration is normal, the subsequent release of LH from the anterior pituitary may not be. A new study by Fernandez and colleagues [56
] targeted the critical period for pituitary differentiation (PND 13) in female rats. Levels of GnRH-induced LH released from pituitary cells in vivo and in vitro were lower than controls in BPA-treated animals. Additionally, BPA-treated animals showed increased GnRH pulse frequency, hypothesized to cause desensitization of the pituitary, thus leading to blunted LH secretion [56
]. Prepubertal exposure to BPA has also been shown to decrease LH pulse frequency and amplitude in gonadally intact ewes [58
]. Similarly, perinatal but not adult exposure to 1.2 mg/kg bw BPA decreased plasma LH levels in adult OVX mice, an observation that also indicates possible interference with hypothalamic organization and emphasizes that exposure sensitivity is heightened during critical developmental periods. Bisphenol-A has also been shown to reduce ERK activation, which may in turn affect GnRH receptor expression [60
]. Reduced GnRH receptor expression in the pituitary could be a mechanism by which LH release is reduced while steroid-induced GnRH activation remains intact. Collectively, these prior results combined with the abnormalities seen in the present study could also be indicative of disrupted steroid-negative feedback.
Although it is still unclear by what mechanism it might occur, our results clearly demonstrate that ovarian cyclicity is compromised by a very brief, neonatal BPA exposure at doses at and above the current EPA reference dose for human exposure. Prior studies examining whether BPA can affect ovarian function have used equivalent or higher doses than those used here, and they have shown that the window of vulnerability may extend beyond the neonatal period [56
]. For example, exposures to 10 mg/kg BPA on PNDs 15–18, 100 μg/kg BPA across PNDs 1–5, or 50 mg/kg BPA on Gestational Days 6–21 have all been shown to result in ovarian and vaginal malformations in mice [63
], including the absence of CLs, cystic ovaries, endometrial hyperplasia, and lack of ESR1 expression in the vagina. Higher doses of BPA (1 or 4 mg per pup) have also been reported to decrease ovarian area occupied by CLs and multiple cystic follicles in rats [66
]. Within that study, however, a dose of 250 μg per pup was not found to alter ovarian morphology, a finding which is inconsistent with our own, but the ovaries were collected approximately 6 wk after vaginal opening (earlier than was done in the present study), so it is possible that abnormalities following low-dose exposure could have manifested later. Although the low dose of BPA accelerated pubertal onset in the present study, most of these animals maintained a regular estrous cycle for 15 wk after DOV, whereas animals exposed to the high dose did not. Our results indicate that lower doses of neonatal BPA exposure may result in reduced ovulatory capacity, although the onset of irregular cycles may take somewhat longer to manifest. This type of effect has also been observed after neonatal low-dose exposure to the phytoestrogen GEN [24
]. Therefore, it is important to observe animals for several weeks beyond pubertal onset when seeking to determine whether an EDC can affect ovarian function. The effect of low-dose BPA on ovarian cyclicity may be more robust if exposure comprises both the gestational and neonatal periods, a condition that is more consistent with human exposure, which is low and lifelong [6
It has been hypothesized that neonatal exposure to EDCs enhances ovulation rate when animals are young, which then results in compromised fertility later in life and induces a premature reproductive senescence [24
]. The intriguing observation that two females exposed to the low (50 μg/kg bw) dose of BPA in the present study had nearly twice as many CLs as control females whereas the rest had fewer is consistent with this hypothesis and requires further investigation. It is possible that among these animals, CL counts were different simply because they were at different points in their estrous cycle when the ovaries were collected. A recent study, however, found that although the morphologic features of CLs change across the cycle, the number of CLs does not [44
Our results also indicate that lower rather than higher doses of BPA have the greatest potential to accelerate pubertal onset, a finding that is counterintuitive but consistent with what has been seen in other studies. An even lower dose (20 μg/kg) administered over Gestational Days 11–17 has also been shown to advance vaginal opening [67
], demonstrating that perturbations of the hormonal milieu in other critical developmental windows besides the neonatal period have the potential to advance puberty. Conversely, higher doses of BPA typically fail to affect pubertal timing, or they delay rather than advance it [64
]. The mechanism by which lower, but not higher, doses of BPA can advance puberty remains poorly understood. It has been hypothesized previously that BPA, like hormones, has a U-shaped or “nonmonotonic” dose-response curve [8
]. Our results are consistent with that hypothesis. The mechanism(s) by which this might occur have not been satisfactorily elucidated but could include stimulation of hormone receptors at low doses but downregulation of hormone receptors at higher doses. (For a critical discussion of nonmonotonic dose curves, see Vandenberg et al. [71
].) It is important to note that although the effect of neonatal exposure to the low dose of BPA on DOV was significant, it was not as robust as neonatal exposure to EB. Neonatal exposure to the ESR1 agonist PPT was also not sufficient to advance pubertal onset to the same degree as EB. This could suggest that strong agonism of both ESR1 and ESR2 is necessary to evoke a maximal response.
It is also possible that BPA alters pubertal timing by an ER-independent mechanism. For example, bw at the time of vaginal opening may influence this endpoint, and could therefore be a confounding factor. Delayed pubertal onset after exposure to 500 mg/kg BPA via maternal dietary exposure during gestation and nursing was most pronounced in the lightest animals [72
]. Other studies in rats and sheep have also found an effect of BPA on body weight at pubertal onset [59
]. Therefore, it could be that vaginal opening in our high-dose (50 mg/kg) animals was later than in our low-dose (50 μg/kg) animals because of a difference in bw. Unfortunately, this possibility was not taken into account; however, we did not note any obvious differences in bw at the time of vaginal opening. Regardless, our data suggest that exposure to BPA during a developmental window that corresponds to late gestation in humans, at levels relevant to human exposure, has the potential to accelerate pubertal onset. The specific mechanisms by which this occurs remain to be determined.
Malformations in the ovary after exposure to EDCs, including BPA, during development have been reported previously, but the mechanism(s) by which they might occur has not been well established [9
]. For this aspect of the present study, the ESR1-selective agonist PPT was employed for comparative purposes. All groups except the control group showed some degree of abnormalities, the characteristics of which were distinctly different between groups. Malformations within the BPA females were dose dependent, with ovaries from the high-dose group showing the most significant adverse effects. Generally, the BPA ovaries more closely resembled those from the PPT than the EB group which, as expected from prior reports [73
], were notably undersized (a qualitative assessment) and showed no sign of folliculogenesis. Unlike the EB group, ovaries from animals in the PPT and high-dose BPA groups were characterized by numerous antral-like cavities, most of which contained either a degenerating or no oocyte. In many cases, these cavities resembled cystic follicles, but some appeared to be large atretic follicles, particularly in the PPT animals (). This observation is consistent with a previous study that also described the presence of large, antral-like cavities in the ovaries of mice exposed to 250 μg/kg bw BPA in utero [74
], and to a more recent study [75
] that observed similar structures in cultured follicles directly exposed to BPA. It should be noted, however, that a different mechanism of action is likely involved in the cell culture study, because the ovarian malformations arose from acute rather than developmental exposure. Finally, none of the PPT females had CLs, an observation that is consistent with what has been reported previously in mice [30
] and suggests that the PPT animals are anovulatory and fail to progress beyond the follicular phase. It appears that agonism of ESR1 during the rodent neonatal period can curtail the capacity of oogenesis to progress into the luteal phase.
Although the ovaries from the animals neonatally treated with PPT and the high dose of BPA share some similar characteristics, they do not necessarily indicate a mechanistic role for ESR1 in the emergence of the BPA-induced effects. Moreover, a mechanistic role for ESR2 in the disruption of ovarian function by BPA cannot be ruled out. Recent studies have reported that ESR2-deficient mice exposed gestationally to 400 ng/day BPA do not display the same abnormalities as BPA-exposed wild-type controls [76
], leading the authors to conclude that BPA exerts its effects in the ovary via ESR2 [76
]. It is important to be mindful, however, that effects resulting from the elimination of ESR2 function across the lifespan may differ from those observed after selectively agonizing or silencing ESR2 during discrete time periods. We and others have shown that postnatal exposure to ESR2-specific agonists can also result in the premature loss of a regular estrous cycle, although not necessarily at the same rate as animals exposed to ESR1-specific agonists [11
]. Therefore, the loss of ovarian cyclicity appears to be possible after postnatal agonism of either of the two major ER subtypes. ESR1 is primarily expressed in the interstitial and thecal cells as early as PND 1, whereas ESR2, expressed in the granulosa cells, is not detectable on PND 1 but is present by PND 5 [79
]. Thus, the BPA exposure in our study was sufficiently long enough for either ER subtype to play a mechanistic role. It is also possible that BPA is acting as an ESR 1 or ESR2 antagonist. Selective antagonists for ESR1 and ESR2 are only now becoming available, so it may soon be possible to directly test how ER-specific antagonism affects ovarian structure and function as part of future studies. These and other types of experiments are needed delineate the relative roles each ER subtype play in the organization and function of the HPG axis, including the ovary.
It is also possible that BPA action on ovarian development and HPG organization may not be limited to ERs. Bisphenol-A is thought to bind to the thyroid hormone receptor, androgen receptor, estrogen-related receptor, and the aryl hydrocarbon receptor [71
], and it may have direct or indirect actions on the ovary through these alternate pathways. For example, estrogen-related receptor gamma (ESRRG) and estrogen-related receptor alpha (ESRRA) expression have been found in human ovarian cells, with increased expression in ovarian cancer cells [83
]. Bisphenol-A can also bind to a membrane-bound form of the ER and a transmembrane ER called G protein-coupled receptor 30 (GPER, formerly called GPR30) [85
]. Emerging evidence now indicates that epigenetic modifications, such as DNA methylation of transposable elements and cis
-acting, imprinting regulatory elements, may also be a potential mechanism by which BPA and other endocrine disruptors affect reproductive function [88
]. The potential for BPA and other endocrine disruptors to modify the epigenome and the health consequences of those disruptions remain largely unexplored.
Interestingly, when hormone primed after ovariectomy, all of the animals except those treated with EB displayed normal sexual receptivity, regardless of whether or not they had lost their estrous cycle, demonstrating that the capacity to display sexual behavior may persist despite impaired reproductive physiology. This observation is consistent with what we have seen before following neonatal exposure to GEN, EQ, PPT, or DPN [11
]. Enhanced lordosis has been reported previously after gestational exposure to 40 μg/kg per day BPA [90
] or a low (5 mg/kg) dose of the chlorinated pesticide chlordecone, a compound which is also classified as an estrogenic EDC [91
]. Our observations are also consistent with a body of literature produced by Gorski [92
], Dohler [93
], and Yanase and Gorski [94
] showing that lordosis behavior can persist after postnatal hormone manipulation, depending on dose and timing of administration. We have yet to determine whether or not these females would display normal sexual behavior if tested with ovaries in place and without hormone priming.
Here, we have shown that neonatal exposure to BPA at or below the LOAEL set by the EPA adversely affects pubertal timing and ovarian function. Our results are consistent with the conclusions of the National Toxicology Program [35
], which stated that there is “some concern for adverse effects of developmental toxicity for fetuses, infants and children.” The National Toxicology Program also concluded that there is “minimal” concern for effects on puberty in females, largely because the mouse literature on this effect is inconsistent, and not enough information has been obtained from rat studies. Therefore, our study adds to the literature demonstrating an effect on sexual maturation in females. Bisphenol-A is known to cross the placenta and pass to infants by lactational transfer [5
]. Blood and amniotic fluid samples obtained from pregnant women reveal that amniotic levels of BPA (8.3–8.7 ng/ml) can be even higher than fetal serum levels (1–2 ng/ml) [97
]. Whether or not the levels of BPA obtained through maternal exposure in humans are sufficient to elicit reproductive deficits remains to be determined. However, as contamination levels and routes of exposure to both naturally occurring and synthetic EDCs differ across populations, determining exactly how they interfere with normal reproductive development warrants further exploration. Our finding that the capacity for GnRH neurons to produce FOS in response to hormone priming is apparently intact in female rats neonatally exposed to BPA suggests that defeminization of the hypothalamus may not be a mechanism by which developmental exposure to BPA can affect DOV, estrous cyclicity, and ovarian morphology. Further studies are needed to better characterize the specific mechanisms through which BPA and similar EDCs disrupt the organization of the HPG axis and impair female reproduction.